14-3-3 targeting peptides for cancer treatment

ABSTRACT

Systems and methods for treatment of squamous cell carcinoma or other cancer utilizing targeting peptides are described. The targeting peptides interact with SCC cells or other cancerous cells to block or interfere with 14-3-3ε heterodimerization or CDC25A binding to 14-3-3ε. A peptide composition embodiment includes, but is not limited to, at least one of a first targeting peptide comprising a structure of Ac-Gln-Arg-Gln-Asn-Ser-(PO32−)-Ala-Pro-Ala-Arg-NH2 (SEQ ID NO: 4) and a second targeting peptide comprising a structure of Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO32−)-Trp-Ala-Gly-NH2 (SEQ ID NO: 5), and a third targeting peptide comprising a modification of or a substituted derivative of the peptide of SEQ ID NO: 4 or SEQ ID NO: 5.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part under 35 U.S.C. § 120of U.S. patent application Ser. No. 16/870,253, filed May 8, 2020, andtitled “14-3-3 TARGETING PEPTIDES FOR CANCER TREATMENT,” which in turnclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Ser. No. 62/845,156, filed May 8, 2019, and titled “14-3-3TARGETING PEPTIDES FOR CANCER TREATMENT.” U.S. Provisional ApplicationSer. No. 62/845,156 and U.S. patent application Ser. No. 16/870,253 areeach herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named“100900144USI1_Sequence_Listing_5.7.2021_ST25.txt”, which is 9.08kilobytes in size (measured in MSWindows®), contains 18 sequences, andwhich was created on May 7, 2021, is contemporaneously filed with thisspecification by electronic submission (using the United States PatentOffice EFS-Web filing system) and is incorporated herein by reference inits entirety.

BACKGROUND

The skin is the largest organ of the body, with an average total area of20 square feet. It serves many important functions including protectionfrom external assaults, sensation of heat and cold, regulation of bodytemperature, water retention, storage and synthesis of vitamin D andexcretion. These functions are supported by the skin's structure, whichis composed of three primary layers: The epidermis, dermis andsubcutaneous. The outermost epidermis and underlying dermis areseparated by a basement membrane, with the dermis resting on asubcutaneous tissue of fat that attaches the skin to the underlyingmuscles and bones. The structure of the skin is reliant upon a closelyregulated process of cell proliferation, differentiation, and death thathelps to preserve not only the structural integrity of the skin, butalso its ability to protect the body from external assaults and maintainbodily homeostasis. As such, any disturbance in the regulation of celldivision or cell death has the potential to lead to cancer or otherpathologies.

SUMMARY

Systems and methods for treatment of squamous cell carcinoma or othercancer utilizing targeting peptides are described. A peptide compositionembodiment includes, but is not limited to, one or more of a firsttargeting peptide comprising a structure of Ac-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro-Ala-Arg-NH₂ (SEQ ID NO: 4), a second targeting peptidecomprising a structure of Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃²⁻)-Trp-Ala-Gly-NH₂ (SEQ ID NO: 5), and a third targeting peptidecomprising a modification of or a substituted derivative of the peptideof SEQ ID NO: 4 or SEQ ID NO: 5.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used as an aid in determining the scope of the claimed subjectmatter.

DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The Detailed Description is described with reference to the accompanyingfigures. In the figures, the use of the same reference numbers indifferent instances in the description and the figures may indicatesimilar or identical items.

FIG. 1A is a dose response curve for a first targeting peptide (ES1P2)with respect to squamous cell carcinoma (SCC) cells in accordance withan example implementation of the present disclosure.

FIG. 1B shows Caspase-3/7 Glo assay results to determine apoptosis forSCC cells treated with vehicle or the first targeting peptide (ES1P2).

FIG. 1C is an immunoblot of immunoprecipitated 14-3-3ε protein fromSCC12B.2 cells treated with vehicle or the first targeting peptide(ES1P2).

FIG. 1D shows Neutral Red Cell Viability assay results of the toxicityin SCC cells of the first targeting peptide (ES1P2) and an agentutilized for treatment of premalignant and malignant skin lesions.

FIG. 1E shows TUNEL (terminal uridine nick-end labelling) assay resultsto detect apoptosis on whole tumor sections from vehicle-treated orES1P2-treated mouse xenograft tumors.

FIG. 1F shows immunofluorescence results for P-Akt (S473) invehicle-treated and in ES1P2-treated SCC xenografts.

FIG. 1G shows immunofluorescence results for Survivin in vehicle-treatedand in ES1P2-treated SCC xenografts.

FIG. 1H shows a chart of tumor volume over time for vehicle-treated SCCcells and ES1P2-treated SCC cells.

FIG. 1I shows a chart of tumor weight following dissection forvehicle-treated SCC cells and ES1P2-treated SCC cells.

FIG. 2A is a dose response curve for a second targeting peptide (pS)with respect to SCC cells in accordance with an example implementationof the present disclosure.

FIG. 2B is a dose response curve for a third targeting peptide (pT) withrespect to SCC cells in accordance with an example implementation of thepresent disclosure.

FIG. 2C shows a Caspase-3/7 Glo assay results to determine apoptosis forSCC cells treated with vehicle, the second targeting peptide (pS), orthe third targeting peptide (pT).

FIG. 2D is an immunoblot of immunoprecipitated 14-3-3ε protein fromSCC12B.2 cells treated with vehicle or the second targeting peptide(pS).

FIG. 2E is an immunoblot of immunoprecipitated 14-3-3ε protein fromSCC12B.2 cells treated with vehicle or the third targeting peptide (pT).

FIG. 3A shows TUNEL assay results to detect apoptosis on whole tumorsections from vehicle-treated, pS-treated, or pT-treated mouse xenografttumors.

FIG. 3B shows immunofluorescence results for P-Akt (S473) invehicle-treated, pS-treated, or pT-treated SCC xenografts.

FIG. 3C shows immunofluorescence results for Survivin invehicle-treated, pS-treated, or pT-treated SCC xenografts.

FIG. 4A shows differential scanning fluorimetry (DSF) results of bindingaffinity of the third targeting peptide to 14-3-3ε protein.

FIG. 4B shows differential scanning fluorimetry (DSF) results of bindingaffinity of a fragment of the third targeting peptide to 14-3-3εprotein.

FIG. 4C shows differential scanning fluorimetry (DSF) results of bindingaffinity of a Tyr substitution to a fragment of the third targetingpeptide to 14-3-3ε protein.

FIG. 4D shows differential scanning fluorimetry (DSF) results of bindingaffinity of the second targeting peptide to 14-3-3ε protein.

FIG. 4E shows differential scanning fluorimetry (DSF) results of bindingaffinity of a fragment of the second targeting peptide to 14-3-3εprotein.

FIG. 4F shows differential scanning fluorimetry (DSF) results of bindingaffinity of a Tyr substitution to a fragment of the second targetingpeptide to 14-3-3ε protein.

FIG. 4G shows differential scanning fluorimetry (DSF) results of bindingaffinity of a Phe substitution to a fragment of the second targetingpeptide to 14-3-3ε protein.

DETAILED DESCRIPTION

Overview

Skin cancer is the most common form of cancer, accounting for 40% of allcases globally. Malignancy is associated with genetic instability thatresults from DNA mutations. In skin cancers, much if not all of thedamage to DNA is a result of chronic exposure to UV radiation. Skincancers can be broadly segregated into two groups, melanoma and the muchmore common non-melanoma skin cancers (NMSC). Basal cell carcinoma (BCC)is the most prevalent form of NMSC making up 80% of all NMSC cases. BCCarises from the keratinocytes of the stratum basale and are slowgrowing, non-life threatening, and very rarely metastasize beyond theoriginal tumor site as shown by a rate of metastasis below 0.1%.Squamous cell carcinoma (SCC) is the second most frequent form of NMSCand can become deadly if unattended.

Contrary to the low metastasis rate of BCCs, SCCs have a 5% chance tometastasize of which 40% will result in death. The location of SCCs candetermine the aggressiveness of the cancer, as primary tumors insun-exposed areas of the skin only have a 5% chance of metastasis whilethose that originate in non-sun-exposed areas of the skin have a 20% ormore rate of metastasis. Additionally, the rate of SCC is upwards of 250times higher in those with a compromised immune system, such as organtransplant recipients, and these SCCs present a much more aggressivephenotype.

Treatments for SCC typically include invasive surgical procedures, suchas surgical excision. Treatments for other NMSCs can have negative sideeffects, including the development of different types of NMSCs. Forexample, vismodegib is a chemotherapy drug used to treat BCC that canlead to the development of SCCs during treatment.

Accordingly, the present disclosure is directed, at least in part, tosystems and methods for treatment of SCCs or other cancers utilizingsynthetic targeting peptides. The targeting peptides target and interactwith specific 14-3-3 proteins that typically regulate protein stability,localization, or activity within cells through binding mechanisms withclient proteins, such as cell cycle regulator cell division cycle 25(CDC25) phosphatases. CDC25 phosphatases (e.g., CDC25A, CDC25B, andCDC25C) and 14-3-3 proteins (e.g., isoforms β, ε, η, σ, θ, γ and ζ) havebeen implicated in various cancers where they often promote celldivision or survival. Surprisingly, research conducted revealed thatCDC25A overexpression or silencing did not impact proliferation in SCCcells, but instead, CDC25A suppressed apoptosis in a manner dependent on14-3-3 and cytoplasmic localization. Both CDC25A and 14-3-3ε activatedAkt, inhibited pro-apoptotic protein Bcl2-associated death promoter(BAD), and increased Survivin, leading to increased SCC cell survival.The targeting peptides of the present disclosure were designed andsynthesized to block 14-3-3ε heterodimerization or CDC25A binding to14-3-3ε. The targeting peptides of the present disclosure successfullydisrupted CDC25A binding to 14-3-3ε and 14-3-3ε heterodimerization,reduced SCC cell viability, increased apoptosis, and decreased activeAkt and Survivin levels, providing a mechanism for skin cancertreatment.

Example Implementations

In embodiments, the targeting peptide includes one or more of a firsttargeting peptide, a second targeting peptide, and a third targetingpeptide to at least one of bind to 14-3-3ε heterodimerization bindingsites or bind to one or more of two 14-3-3 binding sites of CDC25A. Inembodiments, the first targeting peptide includes peptide ES1P2(Trp-Tyr-Trp-Lys-NH₂ (SEQ ID NO: 1)) and has an IC₅₀ of 20.6 μM in SCCcells to reduce 14-3-3ε heterodimerization and increase apoptosis of SCCcells. In embodiments, the second targeting peptide includes peptide pS(phospho-Ser178; Ac-Thr-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro-Arg-Met-Leu-Ser-Ser-Asn-NH₂ (SEQ ID NO: 2)) and has an IC₅₀of 29 μM to induce SCC cell death and block 14-3-3ε binding to CDC25A.In embodiments, the third targeting peptide includes peptide pT(phospho-Thr507 residue; Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃²⁻)-Trp-Ala-Gly-Glu-Lys-Ser-Lys-Arg-NH₂(SEQ ID NO: 3)) and has an IC₅₀of 22.1 μM to induce SCC cell death and block 14-3-3ε binding to CDC25A.

In embodiments, the targeting peptide includes a fragment, a derivative,a modified substitution, or combinations thereof of one or more of thesecond targeting peptide or the third targeting peptide. In embodiments,the targeting peptide includes Ac-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro-Ala-Arg-NH₂ (SEQ ID NO: 4). In embodiments, the targetingpeptide includes Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala-Gly-NH₂ (SEQID NO: 5).

In embodiments, the targeting peptide includes a fragment, a derivative,a modified substitution, or combinations thereof ofAc-Gln-Arg-Gln-Asn-Ser-(PO₃ ²⁻)-Ala-Pro-Ala-Arg-NH₂ (SEQ ID NO: 4)and/or Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala-Gly-NH₂ (SEQ ID NO:5). Example derivatives of Ac-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro-Ala-Arg-NH₂ (SEQ ID NO: 4) are shown in Table 1:

TABLE 1 Derivatives of Ac-Gln-Arg-Gln-Asn-Ser-(PO₃ ²⁻)-Ala-Pro-Ala-Arg-NH₂(SEQ ID NO: 4) SEQ  ID NO Structure SEQ ID Ac-Gln-Arg-Gln-Xaa-Ser-(PO₃ ²⁻)-Ala-Pro- NO: 6 Ala-Arg-NH₂Xaa = Arg, Lys, Orn, Trp, Tyr, Phe, or His SEQ ID Ac-Gln-Arg-Gln-Asn-Ser-(PO₃ ²⁻)-Xaa-Pro- NO: 7 Ala-Arg-NH₂Xaa = Trp, Tyr, Phe, or His SEQ ID  Ac-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro- NO: 8 Xaa-Arg-NH₂ Xaa = Trp, Tyr, Phe, or His SEQ ID Ac-Gln-Arg-Xaa-Asn-Ser-(PO₃ ²⁻)-Ala-Pro- NO: 9 Ala-Arg-NH₂Xaa = Arg, Lys, Orn, Pro, Trp, Tyr, Phe,  or His SEQ ID Ac-Gln-Xaa-Gln-Asn-Ser-(PO₃ ²⁻)-Ala-Pro- NO: 10 Ala-Arg-NH₂Xaa = Lys or Orn SEQ ID  Ac-Xaa-Arg-Gln-Asn-Ser-(PO₃ ²⁻)-Ala-Pro- NO: 11Ala-Arg-NH₂ Xaa = Trp, Tyr, Phe, or His

In some embodiments, the targeting peptide includes an inversomodification, a retro-inverso modification, partial retro-inversomodification, or a retro modification of Ac-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro-Ala-Arg-NH₂ (SEQ ID NO: 4) or the derivatives shown inTable 1. Inverso modifications include peptides made up of the D-aminoacid enantiomers of L-amino acids (e.g., Ac-gln-arg-gln-asn-ser-(PO₃²⁻)-ala-pro-ala-arg-NH₂). Retro-inverso modifications include peptidesmade up of the of the D-amino acid enantiomers of L-amino acid residuesassembled in reverse order of the L-amino acid sequence (e.g.,Ac-arg-ala-pro-ala-ser-(PO₃ ²⁻)-asn-gln-arg-gln-NH₂). Partialretro-inverso peptides include modifications in which only part of thesequence is reversed and replaced with enantiomeric amino acid residues.Retro peptides include modifications made up of L-amino acid residuesassembled in reverse order. In some embodiments, the targeting peptideincludes a combination(s) of the substitutions described in Table 1.

Example derivatives of Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃²⁻)-Trp-Ala-Gly-NH₂ (SEQ ID NO: 5) are shown in Table 2:

TABLE 2 Derivatives of Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala-Gly-NH₂ (SEQ ID NO: 5) SEQ  ID NO Structure SEQ ID Ac-Arg-Thr-Lys-Ser-Xaa-Thr(PO₃ ²⁻)-Trp-Ala- NO: 12 Gly-NH₂Xaa = Lys, Orn, Trp, Tyr, Phe, or His SEQ ID Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Xaa-Ala- NO: 13 Gly-NH₂Xaa = Ala, Tyr, Phe, or His SEQ ID  Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃²⁻)-Trp-Pro- NO: 14 Gly-NH₂ SEQ ID  Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃²⁻)-Trp-Ala- NO: 15 Xaa-NH₂ Xaa = Trp, Tyr, Phe, or His SEQ ID Ac-Arg-Thr-Lys-Xaa-Arg-Thr(PO₃ ²⁻)-Trp-Ala- NO: 16 Gly-NH₂Xaa = Arg, Lys, Orn, Pro, Trp, Tyr, Phe,  or His SEQ ID Ac-Arg-Thr-Xaa-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala- NO: 17 Gly-NH₂Xaa = Arg or Orn SEQ ID  Ac-Xaa-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala-NO: 18 Gly-NH₂ Xaa = Trp, Tyr, Phe, or His

In some embodiments, the targeting peptide includes an inversomodification (e.g., Ac-arg-thr-lys-ser-arg-thr(PO₃ ²⁻)-trp-ala-Gly-NH₂,a retro-inverso modification (e.g., Ac-Gly-ala-trp-thr(PO₃²⁻)-arg-ser-lys-thr-arg-NH₂), a partial retro-inverso modification, or aretro modification of Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala-Gly-NH₂(SEQ ID NO: 5) or the derivatives shown in Table 2. In some embodiments,the targeting peptide includes a combination(s) of the substitutionsdescribed in Table 2.

In embodiments, one or more targeting peptides are included in a peptidecomposition having activity against squamous cell carcinoma survival.For example, the peptide composition can include one or more of ES1P2,pS, and pT. The peptide composition can include a pharmaceuticallyacceptable carrier to facilitate delivery of the peptide composition toone or more regions of an individual subject containing SCC cells. Forexample, the pharmaceutically acceptable carrier is suitable foradministration via at least one of administration via injection, aerosoladministration, administration via inhalation, oral administration,systemic IV application, ocular administration, and rectaladministration.

The one or more targeting peptides can include a peptide having theformula X—R¹—R²—R³—R⁴—Y, where X represents hydrogen, acetyl, orpropionyl group, R¹ through R⁴ represent all possible combinations of 19standard L-amino or D-amino acid residues (excluding Cys): Ala, Arg,Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, Val, and Y represents —OH, amide, methylamide or ethylamidegroups. The one or more targeting peptides can include a peptide havingthe formula X—R¹R²—R³—R⁴—R⁵—R⁶—Y, where X represents hydrogen, acetyl,or propionyl group, R¹ through R⁶ represent all possible combinations of19 standard L-amino or D-amino acid residues (excluding Cys): Ala, Arg,Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, Val, and Y represents —OH, amide, methylamide or ethylamidegroups. The one or more targeting peptides can include peptide ES1P2(Trp-Tyr-Trp-Lys-NH₂ (SEQ ID NO: 1)) with a connection at a carboxyterminal end to a hex-Gly spacer connected to peptide pS(phospho-Ser178; Ac-Thr-Gln-Arg-Gln-Asn-Ser-(PO₃²⁻)-Ala-Pro-Arg-Met-Leu-Ser-Ser-Asn-NH₂ (SEQ ID NO: 2)). The one or moretargeting peptides can include peptide ES1P2 (Trp-Tyr-Trp-Lys-NH₂ (SEQID NO: 1)) with a connection at a carboxy terminal end to a hex-Glyspacer connected to peptide pT (phospho-Thr507 residue;Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Trp-Ala-Gly-Glu-Lys-Ser-Lys-Arg-NH₂(SEQ ID NO: 3)).

The one or more targeting peptides can include a peptide having theformula X—R¹—R²—R³—R⁴-Ser-(PO₃ ²⁻)—R⁵-Pro-R⁶-Arg-Y, where X representshydrogen, acetyl or propionyl group, R¹ represents an L or D-amino acidresidue selected from the group consisting of: Gln, Trp, Tyr, Phe, His,R² represents an L or D-amino acid residue selected from the groupconsisting of: Arg, Lys, Orn, R³ represents an L or D-amino acid residueselected from the group consisting of: Gln, Arg, Lys, Orn, Pro, Trp,Tyr, Phe, His, R⁴ represents an L or D-amino acid residue selected fromthe group consisting of: Asn, Arg, Lys, Orn, Trp, Tyr, Phe, His, R⁵represents an L or D-amino acid residue selected from the groupconsisting of: Ala, Trp, Tyr, Phe, His, R⁶ represents an L or D-aminoacid residue selected from the group consisting of: Ala, Trp, Tyr, Phe,His, and Y represents —OH, amide, methylamide, or ethylamide groups.

The one or more targeting peptides can include a peptide having theformula X—R¹-Thr-R²—R³—R⁴-Thr(PO₃ ²⁻)—R⁵—R⁶—R⁷—Y, where X representshydrogen, acetyl or propionyl group, R¹ represents an L or D-amino acidresidue selected from the group consisting of: Arg, Trp, Tyr, Phe, His,R² represents an L or D-amino acid residue selected from the groupconsisting of: Lys, Arg, Orn, R³ represents an L or D-amino acid residueselected from the group consisting of: Ser, Arg, Lys, Orn, Pro, Trp,Tyr, Phe, His, R⁴ represents an L or D-amino acid residue selected fromthe group consisting of: Arg, Lys, Orn, Trp, Tyr, Phe, His, R⁵represents an L or D-amino acid residue selected from the groupconsisting of: Trp, Ala, Tyr, Phe, His, R⁶ represents an L or D-aminoacid residue selected from the group consisting of: Ala, Pro, R⁷represents an L-Glycine or an L or D-amino acid residue selected fromthe group consisting of: Trp, Tyr, Phe, His, and Y represents —OH,amide, methylamide, or ethylamide groups.

In embodiments, the peptide composition can include one or moreadditional ingredients including, but not limited to, pharmaceuticalfiller ingredients.

In some embodiments, the peptide compositions described herein areadapted for topical application. Such compositions can include thepeptide conjugated together with a carrier to facilitate topicalapplication. Such a composition may be, for example, in the form ofsolutions, suspensions, emulsions, lotions, creams, microemulsions,nanoemulsions, emulgels, gels, and the like. Compositions describedherein may also extend to patches and plasters for application to skinand incorporating peptides in a form such that it will be released intothe skin.

In some embodiments, peptide compositions described herein includepharmaceutically and/or dermatologically acceptable excipientsincluding, but not limited to, one or more of carriers, emulsifiers,coemulsifiers, permeation or penetration enhancers, solvents,co-solvents, emollients, antioxidants, preservatives, buffering agents,gelling or thickening agents, polymers, surfactants, soothing agents, pHmodifiers, solubilizers, humectants, emollients, moisturizers, oilybases, and the like.

The term “carrier” or “vehicle” denotes organic or inorganicingredients, natural or synthetic, with which an active ingredient iscombined to facilitate application of a composition. Examples ofcarriers include, but not limited to, water, acetone, alone or incombination with materials such as silicone fluids. In certainembodiments, the carrier can comprise, in addition to water,water-immiscible substances such as any pharmaceutically acceptablefatty esters of natural fatty acids, triglycerides of animal orvegetable, medium chain triglycerides, mixtures of mono-, di- and/ortriglycerides, waxes, hydrogenated vegetable oils, and mixtures thereof.

Examples of emulsifiers include, but not limited to, disodium cocoamphodiacetate, oxyethylenated glyceryl cocoate (7 EO), PEG-20 hexadecenylsuccinate, PEG-15 stearyl ether, ricinoleic monoethanolamidemonosulfosuccinate salts, oxyethylenated hydrogenated ricinoleictriglyceride containing 60 ethylene oxide units such as the productsmarketed by BASF under the trademarks CREMOPHOR® RH 60 or CREMOPHOr® RH40 (polyoxyl 40 hydrogenated castor oil), polymers such as poloxamers,which are block copolymers of ethylene oxide and propylene oxide, andthe nonsolid fatty substances at room temperature (that is to say, attemperatures ranging from about 20 to 35° C.) such as sesame oil, sweetalmond oil, apricot stone oil, sunflower oil, octoxyglyceryl palmitate(or 2-ethylhexyl glyceryl ether palmitate), octoxyglyceryl behenate (or2-ethylhexyl glyceryl ether behenate), dioctyl adipate, and tartrates ofbranched dialcohols. Sorbitan fatty acid esters are a series of mixturesof partial esters of sorbitol and its mono- and dianhydrides with fattyacids. Sorbitan esters include products marketed as ARLACEL® 20, ARLACEL40, ARLACEL 60, ARLACEL 80, ARLACEL83, ARLACEL 85, ARLACEL 987, ARLACELC, PEG-6 stearate and glycol stearate and PEG-32 stearate (TEFOSE® 63),and PEG-6 stearate and PEG-32 stearate (TEFOSE® 1500), glyceryl stearateand PEG 100 stearate (TEFOSE® 165) and any mixtures thereof.Polyethylene glycol ethers of stearic acid are in another group ofemulsifiers that can be used in the emulsions. Examples of polyethyleneglycol ethers of stearic acid include, but not limited to, steareth-2,steareth-4, steareth-6, steareth-7, steareth-10, steareth-11,steareth-13, steareth-15, steareth-20, polyethylene glycol ethers ofstearyl alcohol (steareth 21), and any mixtures thereof. Otheremulsifiers include sodium lauryl sulphate, cetyl trialkyl ammoniumbromide, polyoxyethylene sorbitan fatty acid esters, and any mixturesthereof.

Nonionic emulsifiers include those that can be broadly defined ascondensation products of long chain alcohols, e.g., C8-30 alcohols, withsugar or starch polymers, i.e., glycosides. Various sugars include, butnot limited to, glucose, fructose, mannose, and galactose, and variouslong chain alcohols include, but are not limited to, decyl alcohol,cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, oleylalcohol, and any mixtures thereof. Other useful nonionic emulsifiersinclude condensation products of alkylene oxides with fatty acids suchas alkylene oxide esters of fatty acids. Other nonionic surfactants arethe condensation products of alkylene oxides with 2 moles of fatty acidssuch as alkylene oxide diesters of fatty acids.

Emulsifiers can also include any of a wide variety of cationic, anionic,zwitterionic, and amphoteric surfactants that are known in the art.Examples of anionic emulsifiers include, but are not limited to, alkylisethionates, alkyl and alkyl ether sulfates and salts thereof, alkyland alkenyl ether phosphates and salts thereof, alkyl methyl taurates,and soaps (e.g., alkali metal salts and sodium or potassium salts) offatty acids. Examples of amphoteric and zwitterionic emulsifiers includethose which are broadly described as derivatives of aliphatic secondaryand tertiary amines in which the aliphatic radical can be straight orbranched chain, wherein one of the aliphatic substituents contains fromabout 8 to about 22 carbon atoms and one contains an anionic watersolubilizing group, e.g., carboxy, sulfonate, sulfate, phosphate, orphosphonate. Specific examples include, but not limited to, alkyliminoacetates, iminodialkanoates and aminoalkanoates, imidazolinium andammonium derivatives. Other suitable amphoteric and zwitterionicemulsifiers include betaines, sultaines, hydroxysultaines, alkylsarcosinates, and alkanoyl sarcosinates.

Silicone emulsifiers can include organically modified organopolysiloxanes, sometimes called silicone surfactants. Useful siliconeemulsifiers can include dimethicone copolyols. These materials arepolydimethyl siloxanes, which have been modified to include polyetherside chains such as polyethylene oxide chains, polypropylene oxidechains, mixtures of these chains, and polyether chains containingmoieties derived from both ethylene oxide and propylene oxide.

Co-emulsifiers include, but not limited to, polyoxylglycerides such asoleoyl macrogolglycerides (LABRAFIL® M 1944CS), linoleoylmacrogolglycerides (LABRAFIL® M 2125CS), caprylocaproylmacrogolglycerides (LABRASOL®), cetyl alcohol (and) ceteth-20 (and)steareth-20 (EMULCIRE™ 61 WL 2659), glyceryl stearate (and) PEG-75stearate (GELOT® 64), d-alpha tocopheryl polyethylene glycol 1000succinate (TPGS) and any mixtures thereof.

The term “solvent” refers to components that aid in the dissolution ofthe drug in the formulation. Solvents serve to maintain a solution ofthe drug in the composition. Some solvents can also enhance percutaneouspenetration of drug and/or act as humectants. Solvents that can be usedin the present peptide compositions can include water-immisciblesubstances such as fatty esters of natural fatty acids, triglycerides ofanimal or vegetable, medium chain triglycerides, mixtures of mono-, di-and/or triglycerides, waxes, hydrogenated vegetable oils, and mixturesthereof. Some specific examples include, but not limited to, castor oil,isopropyl myristate, dimethyl isosorbide, oleyl alcohol, labrafil,labrasol, medium chain triglyceride, diethyl sebacate, lanolin oil,citrate triisocetyl triglycerides having 10-18 carbon atoms,caprylic/capric triglycerides, coconut oil, corn oil, cottonseed oil,linseed oil, oil of mink, olive oil, palm oil, sunflower oil, nut oil,saturated paraffin oils, mineral oils, vegetable oils or glycerides, andthe like. Solvent can also be selected from the group comprisingmonoalkyl ether of diethylene glycol such as diethylene glycolmonomethyl ether, diethylene glycol monoethyl ether or mixtures thereof.

The term “emollients” refers to substances that soften and soothe theskin. They can be used to prevent dryness and scaling of the skin.Examples of emollients that can be used in the present peptidecompositions include, but not limited to, oils of natural origin such asalmond oil, coconut oil, olive oil, palm oil, peanut oil and the like,fatty acids such as lauric acid, myristic acid, palmitic acid, andstearic acid, monohydric alcohol esters of the fatty acids such as ethyllaurate, isopropyl laurate, ethyl myristate, n-propyl myristate,isopropyl myristate, ethyl palmitate, isopropyl palmitate, methylpalmitate, methyl stearate, ethyl stearate, isopropyl stearate, butylstearate, isobutyl stearate, amyl stearate, and isoamyl stearate,glycols such as ethylene glycol, diethylene glycol, polyethylene glycol,branched aliphatic alcohols such as lauryl alcohol, myristyl alcohol,and stearyl alcohol, or mixtures thereof. Exemplary emollients includecaprylic/capric triglyerides, castor oil, ceteareth-20, ceteareth-30,cetearyl alcohol, ceteth 20, cetostearyl alcohol, cetyl alcohol, cetylstearyl alcohol, cocoa butter, diisopropyl adipate, glycerin, gycerylmonooleate, glyceryl monostearate, glyceryl stearate, isopropylmyristate, isopropyl palmitate, lanolin, lanolin alcohol, hydrogenatedlanolin, liquid paraffins, linoleic acid, mineral oil, oleic acid, whitepetrolatum, polyethylene glycol, polyoxyethylene glycol fatty alcoholethers, silicones and mixtures thereof.

Silicones are typically organically modified organopoly siloxanes,sometimes called silicone surfactants. Useful polysiloxane or siliconeemollients include, but not limited to, polysiloxane polymer,dimethicone copolyols, cyclomethicones. These materials are polydimethylsiloxanes, which have been modified to include polyether side chainssuch as polyethylene oxide chains, polypropylene oxide chains, mixturesof these chains, and polyether chains containing moieties derived fromboth ethylene oxide and propylene oxide.

The term “antioxidants” are substances which inhibit oxidation orsuppress reactions promoted by oxygen or peroxides. Antioxidants,especially lipid-soluble antioxidants, can be absorbed into the cellularmembrane to neutralize oxygen radicals and thereby protect the membrane.Suitable antioxidants that can be used in the present peptidecompositions include, but not limited to, ascorbic acid (vitamin C),glutathione, lipoic acid, uric acid, sorbic acid, carotenes,α-tocopherol (vitamin E), TPGS, ubiquinol, butylated hydroxyanisole,butylated hydroxytoluene, sodium benzoate, propyl gallate (PG, E310),and tertiary-butylhydroquinone.

The term “preservative” refers to a natural or synthetic chemical thatprevents the decomposition of the composition by microbial growth or byundesirable chemical changes. Preservatives can be incorporated into apeptide composition for protecting against the growth of potentiallyharmful microorganisms. While microorganisms tend to grow in an aqueousphase and can also reside in a hydrophobic or oil phase. Examples ofpreservatives that can be used in the present peptide compositionsinclude, but are not limited to, methylparaben, propylparaben, benzylalcohol, chlorocresol, benzalkonium chloride, cetrimonium chloride,sodium edetate, boric acid, sorbic acid, or any mixtures thereof.

The term “thickening agents” or “gelling agents” are used to givebulkiness to the peptide composition. Examples of thickening agents orgelling agents that can be used in the present peptide compositionsinclude, but not are limited to: carbomers, polyethylene glycols,acrylate polymers, methacrylate polymers, polyvinylpyrrolidones,copolymers based on butyl methacrylate and methyl methacrylate povidone,vinyl acetates, polyvinyl acetates, celluloses, gums, alginates,cellulose acetate phthalates, cellulose acetate butyrates, hydroxypropylmethyl cellulose phthalates, and the like. Examples include CARBOPOL®products, PEG 400, EUDRAGIT® 100, EUDRAGIT® RSPO, EUDRAGIT® RLPO,EUDRAGIT® ND40, PLASDONE®, copolymers based on butyl methacrylate andmethyl methacrylate (PLASTOID® B), alkyl celluloses such as ethylcelluloses and methyl celluloses, hydroxyalkyl celluloses such ashydroxyethyl cellulose and hydroxypropyl cellulose, hydroxyalkyl alkylcelluloses such as hydroxypropyl methyl celluloses and hydroxybutylmethyl celluloses, gums such as xanthan gum, tragacanth, guar gum,locust bean gum, acacia, and the like.

In an embodiment, the thickening agents can include non-polymericthickening agents, such as fatty alcohols. Examples of fatty alcoholsinclude, but are not limited to: cetyl alcohol, paraffin, stearylalcohol, white wax, wax cetyl esters, microcrystalline wax, anionicemulsifying wax, nonionic emulsifying wax, yellow wax, castor oil,ceresin, cetostearyl alcohol, cyclomethicone, glyceryl behenate,hectorite, myristyl alcohol, cetylstearyl alcohol, triolein, andlanolin. Other thickening agents or gelling agents or polymers that areuseful in the present peptide compositions include, but not limited to,polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyglycolides,polysiloxanes, polyurethanes and copolymers thereof, cellulose ethers,cellulose esters, nitrocelluloses, polymers of acrylic and methacrylicesters, cellulose acetates, cellulose propionates, cellulose acetatebutyrates, cellulose acetate phthalates, carboxylethyl celluloses,cellulose triacetates, cellulose sulphate sodium salts, poly(methylethacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylenes,polypropylenes, poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate),poly(vinyl chloride), polystyrenes, and the like, including theirmixtures thereof.

Examples of other useful polymers that can act as thickening agents orgelling agents include, but not limited to, synthetic polymers, such aspolymers of lactic acid and glycolic acid, polyanhydrides, poly(orthoester), polyurethanes, poly(butyric acid), poly(valeric acid),poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide),poly(lactide-co-caprolactone), and natural polymers such as alginate andother polysaccharides that include but not limited to arabinans,fructans, fucans, galactans, galacturonans, glucans, mannans, xylans(such as, for example, inulin), levan, fucoidan, carrageenan,galactocarolose, pectic acid, pectin, amylose, pullulan, glycogen,amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratan,chondroitan, dermatan, hyaluronic acid, alginic acid, xanthan gum,starches, and various other natural homopolymers and heteropolymers,such as those containing one or more of aldoses, ketoses, acids oramines, erythrose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof, and including dextran and cellulose, collagen,albumin and other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof.

In some embodiments, the peptide compositions can further include one ormore penetration enhancers. Penetration enhancers used in the topicalcompositions can provide higher skin layer retention and lower systemicexposure by avoiding the drug entering into systemic circulation, thistendency of the penetration enhancers provides skin depot compositions.

In some embodiments, the peptide composition can further include one ormore antioxidant, preservative, humectant, or plasticizer.

It is contemplated that some of the excipient substances described abovecan have more than one function in a formulation. For example, asubstance can be both a solvent and a penetration enhancer, or both asolvent and a carrier. The categorizations of materials described aboveare not to be construed as limiting or restricting in any manner.

Implementations of example targeting peptides are described below withresearch and methodologies following.

Targeting Peptide to Target 14-3-3 Dimerization to Reduce SCC CellViability

In embodiments, the targeting peptide includes a synthesizedtetrapeptide, ES1P2 (Trp-Tyr-Trp-Lys-NH₂ (SEQ ID NO: 1)), configured toreduce 14-3-3ε dimerization and increase SCC apoptotic cell death.14-3-3 proteins can function as oncogenes by promoting tumor-likecharacteristics such as cell survival, invasion, and proliferation,where 14-3-3 proteins are required for CDC25A-mediated suppression ofapoptosis in skin cancers. Specifically, the 14-3-3ε isoform facilitatesthe activation of anti-apoptotic signaling pathways via Akt/BAD/Survivinsignaling in SCC cells, where 14-3-3ε stabilizes Akt protein andsequesters Survivin into the cytoplasm of SCC cells. 14-3-3εheterodimerization with other 14-3-3 isoforms is required for aspects ofits activity. In SCC cells, 14-3-3ε preferentially forms heterodimerswith 14-3-3γ and -ζ, and targeting 14-3-3ε heterodimerization with thetargeting peptide ES1P2 reduced 14-3-3ε dimerization and increased SCCapoptotic cell death. Without wishing to be bound by any particulartheory, ES1P2 targets N-terminal residues unique to 14-3-3ε to bind andreduce 14-3-3ε dimerization. FIG. 1A shows an example dose responsecurve for the targeting peptide ES1P2 with respect to SCC cells(SCC12B.2 cells) with an IC₅₀ of 20.6 μM FIG. 1B shows an exampleCaspase-3/7 Glo assay (N=5) to determine apoptosis for SCC cells(SCC12B.2 cells) treated with vehicle or the targeting peptide ES1P2 (20μM). The targeting peptide ES1P2 reduced 14-3-3ε heterodimerization,killed SCC cells but not normal keratinocytes, and decreased tumorgrowth.

FIG. 1C shows an example immunoblot of immunoprecipitated 14-3-3εprotein from SCC12B.2 cells treated with vehicle (Tris buffer, pH 7.5)or ES1P2 (20 μM) for 48 hours. The control (−) included an equal numberof cells incubated with IgG isotype control antibody. The immunoblotresults indicate that ES1P2 reduces 14-3 3ε heterodimerization andinduces SCC cell death by increasing apoptosis to a statisticallysignificant degree (e.g., Student's two-tailed t-test, P≤0.05).Referring to FIG. 1A (dashed line) ES1P2 was nontoxic to normalkeratinocytes. Without wishing to be bound by any particular theory, thealtered localization of 14-3-3ε in SCC (cytoplasm) compared to normalskin (nuclear) can play a role in the ability of ES1P2 to induce SCCcell death while being nontoxic to normal keratinocytes.

In implementations, the targeting peptide ES1P2 exhibited greatertoxicity in SCC cells as compared to the toxicity in SCC cells by anagent utilized for treatment of premalignant and malignant skin lesions,5-Fluorouracil (5-FU). For example, FIG. 1D shows a comparative examplevia Neutral Red Cell Viability assay of the toxicity in SCC cells ofES1P2 and 5-FU, where ES1P2 alone was more effective in killing SCCcells (20 μM; 62% alive) than 5-FU alone (2004; 71.5% alive).

In implementations, the targeting peptide ES1P2 decreased P-Akt (S473)and Survivin levels in ES1P2-treated SCC xenografts and reduced tumorvolume in long-term treatments compared to vehicle-treated SCCxenografts. For example, FIG. 1E shows TUNEL (terminal uridine nick-endlabelling) assays to detect apoptosis (FITC, green nuclei) on wholetumor sections from vehicle or ES1P2-treated mouse xenograft tumors withDAPI-labeled nuclei (blue) (scale bar=50 μm). The xenografts wereestablished with a human skin cancer cell line and were treatedintratumorally with either vehicle or 2.5 nmol of ES1P2. After 2 dailytreatments, the number of TUNEL-positive SCC cells more than doubledcompared to vehicle treated. Immunofluorescence revealed a decrease inP-Akt (S473) and Survivin levels in ES1P2-treated SCC xenografts. Forexample, FIG. 1F shows immunofluorescence results for P-Akt (S473) invehicle-treated and in ES1P2-treated SCC xenografts and FIG. 1G showsimmunofluorescence results for Survivin in vehicle-treated and inES1P2-treated SCC xenografts. The targeting peptide ES1P2 also reducedSCC growth by approximately one-third as compared to vehicle-treated SCCcells following daily treatments of the SCC xenografts. For example,FIG. 1H shows a chart of tumor volume over time for vehicle-treated SCCcells and ES1P2-treated SCC cells and FIG. 1I shows a chart of tumorweight following dissection, which provides an example of in vivoapplication.

14-3-3ε promotes anti-apoptotic signaling in SCC cells and is criticalfor tumor development and progression in mouse skin. The targeting of14-3-3ε heterodimerization with the targeting peptide ES1P2 effectivelykilled skin cancer cells and reduced tumor growth through targeting14-3-3ε and reducing the ability of 14-3-3ε to form dimers.

Targeting Peptide to Target 14-3-3 Binding Sites of CDC25A to Reduce SCCCell Viability

In embodiments, the targeting peptide includes one or more syntheticphospho-peptide fragments to target one or two of the binding sites ofCDC25A to 14-3-3ε. The phospho-peptide fragments can include, but arenot limited to, peptide pS (phospho-Ser178;Ac-Thr-Gln-Arg-Gln-Asn-Ser-(PO₃ ²⁻)-Ala-Pro-Arg-Met-Leu-Ser-Ser-Asn-NH₂(SEQ ID NO: 2)) and peptide pT (phospho-Thr507 residue;Ac-Arg-Thr-Lys-Ser-Arg-Thr(PO₃²⁻)-Trp-Ala-Gly-Glu-Lys-Ser-Lys-Arg-NH₂(SEQ ID NO: 3)) to block 14-3-3εbinding to CDC25A and induce SCC cell death. SCC xenografted tumorstreated with peptide pS and peptide pT displayed increased apoptoticcell death and decreased pro-survival P-Akt (S473) and Survivin. Invivo, mouse xenograft tumors treated with both peptide pS and peptide pTdisplayed decreased volume, increased apoptotic activity, and reducedP-Akt (S473) and Survivin expression in SCC cells compared tovehicle-treated tumors.

Without wishing to be bound by any particular theory, peptide pS targetsa first binding site of CDC25A (Ser178) and peptide pT targets a secondbinding site of CDC25A (Thr507) to block or otherwise inhibitinteractions between CDC25A and 14-3-3ε to decrease SCC cell survival.Referring to FIG. 2A, an example dose response curve is shown for thetargeting peptide pS with respect to SCC cells (SCC12B.2 cells) fortwelve samples, with an IC₅₀ of 29 μM. Referring to FIG. 2B, an exampledose response curve is shown for the targeting peptide pT with respectto SCC cells (SCC12B.2 cells) for twelve samples, with an IC₅₀ of 22.6μM. FIG. 2C shows an example Caspase-3/7 Glo assay (N=5) to determineapoptosis for SCC cells (SCC12B.2 cells) treated with vehicle, thetargeting peptide pS (30 μM), or the targeting peptide pT (20 μM) for 24hours. Each of the targeting peptides (pS and pT) decreased SCC cellviability by increased apoptosis.

FIG. 2D shows an example immunoblot of immunoprecipitated 14-3-3εprotein from SCC12B.2 cells treated with vehicle (Tris buffer, pH 7.5)or pS (30 μM) for 48 hours. The control (−) included an equal number ofcells incubated with IgG isotype control antibody. The immunoblotresults indicate that the targeting peptide pS decreases the amount ofCDC25A present on the immunoblots when compared to immunoprecipitationsfrom vehicle treated cells to a statistically significant degree (e.g.,Student's two-tailed t-test, P≤0.05). FIG. 2E shows an exampleimmunoblot of immunoprecipitated 14-3-3ε protein from SCC12B.2 cellstreated with vehicle (Tris buffer, pH 7.5) or pT (2004) for 48 hours.The control (−) included an equal number of cells incubated with IgGisotype control antibody. The immunoblot results indicate that thetargeting peptide pT decreases the amount of CDC25A present on theimmunoblots when compared to immunoprecipitations from vehicle treatedcells to a statistically significant degree (e.g., Student's two-tailedt-test, P≤0.05). In implementations, pS-treated samples showed less14-3-3ε precipitation as compared to pT-treated samples.

In implementations, the targeting peptides pS and pT each decreasedSurvivin and active Akt levels in targeting peptide-treated SCCxenografts as compared to vehicle-treated SCC xenografts. For example,FIG. 3A shows TUNEL (terminal uridine nick-end labelling) assays todetect apoptosis (FITC, green nuclei) on whole tumor sections from mousexenograft tumors treated with vehicle, pS, or pT with DAPI-labelednuclei (blue) (scale bar=50 μm). The xenografts were established withimmunocompromised NCG mice injected with 5×10⁵ SCC13 cells. After 2daily treatments, the number of TUNEL-positive SCC cells increased by25% and 51% in pS and pT treated tumors, respectively, when compared tovehicle treated tumors. Immunofluorescence revealed a decrease in P-Akt(S473) and Survivin levels in ES1P2-treated SCC xenografts. For example,FIG. 3B shows immunofluorescence results for P-Akt (S473) invehicle-treated, pS-treated, and pT-treated SCC xenografts and FIG. 3Cshows immunofluorescence results for Survivin in vehicle-treated,pS-treated, and pT-treated SCC xenografts.

CDC25A associates with 14-3-3ε, 14-3-3γ and 14-3-3ζ in SCC cells. CDC25Ainhibits apoptosis by promoting the activation of Akt, inhibition ofBAD, and an increase in Survivin, the same pathway that is regulated by14-3-3ε. Treatment of SCC cells with the targeting peptides pS and pTreduced the interaction of 14-3-3ε with CDC25A, decreased SCC cellviability and increased apoptosis both in vitro and in vivo for eachtargeting peptide.

Methods and materials used in example diagnostic implementations of theabove-described targeting peptides (ES1P2, pS, pT) are provided below.

Methods and Materials—ES1P2 Targeting Peptide Implementations

Cell Culture

Human skin cancer cell lines (SCC12B.2, SCC13, SRB12) were maintained inDulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, Calif.,USA) supplemented with 1% penicillin (10 000 U/ml)-streptomycin (10 000μg/ml) (PenStrep) (Invitrogen) and 10% fetal bovine serum (GeminiBio-Products, Sacramento, Calif., USA) at 37° C. and 5% CO₂. Normalhuman epidermal keratinocytes (nHEK) (Thermo Scientific, Waltham, Mass.,USA) isolated from neonatal foreskin were cultured in EpiLife medium(Invitrogen) supplemented with human keratinocyte growth supplement(Invitrogen) and 1% PenStrep (Invitrogen). Cells were transfected withcontrol pcDNA or pcDNA3.1-HA-14-3-3ε (Addgene plasmid #48797;http://n2t.net/addgene:48797; RRID:Addgene_48797) usingLipofectamine/Plus reagents (Life Technologies, Carlsbad, Calif., USA)or with siRNA targeting 14-3-3ε (Dharmacon, Lafayette, Colo., USA),14-3-3γ (Dharmacon), 14-3-3ζ, (Dharmacon), Survivin (Cell Signaling,Danvers, Mass., USA), Akt (Dharmacon) or control siRNA (Santa CruzBiotechnology, Dallas, Tex., USA) using siQuest transfection reagents(Mirus Bio, Madison, Wis., USA) or treated with Akt inhibitor GSK690693(Tocris Bioscience, Bristol, United Kingdom) or cell cycle inhibitor5-Fluorouracil (5-FU) (Thermo Fisher) resuspended in DMSO.

Immunofluorescence and Immunohistochemistry

Immunostaining was performed using antibodies recognizing 14-3-3ε (CellSignaling), 14-3-3γ (Cell Signaling), 14-3-3ζ (Cell Signaling), Survivin(Cell Signaling), P-Akt (S473) (Thermo Fisher), Keratin 5 (BioLegend,San Diego, Calif., USA), Keratin 1 (BioLegend), Loricrin (NovusBiologicals, Centennial, Colo., USA) or Ki67 (Abcam, Cambridge, UK).Immunohistochemistry was performed using horseradishperoxidase-conjugated secondary antibody (IgG rabbit) (Cell Signaling)with a hematoxylin (Sigma Aldrich, St. Louis, Mo., USA) counterstain.

Immunofluorescence was performed using an alexa-fluor488-conjugatedsecondary antibody (IgG mouse or rabbit) (Invitrogen) with4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, Calif.,USA) to identify nuclei. Cellular localization of immunohistochemistrysignal was determined in a double-blinded manner. Slides weredigitalized and fluorescence intensity was measured using Image analysiswas performed using the Olympus VS120 Slide Scanner and Olyvia AnalysisSoftware, respectively (Olympus, Tokyo, Japan).

Co-Immunoprecipitation

Co-Immunoprecipitation was performed using a Dynabeads Protein GImmunoprecipitation kit (Life Technologies, Carlsbad, Calif., USA).Beads were coupled to antibodies targeting 14-3-3ε (Fisher Scientific,Waltham, Mass., USA) or Akt (Thermo Fisher) at a ratio of 7 μg ofantibody for every 1 mg of beads according to the manufacturersprotocol. SCC12B.2 cells were treated with vehicle (20 mM Tris buffer,pH 7.5) or fresh ES1P2 targeting peptide daily at the IC₅₀ concentrationfor 48 hours in Opti-MEM I Reduced Serum Medium (Invitrogen) andcollected by trypsinization. One hundred mg of cells were lysed in 1×IPbuffer supplemented with 100 mM sodium chloride, 2 mM dithiothreitol, 1mM magnesium chloride and protease (Roche Life Scientific, Penzberg,Germany) and phosphatase inhibitors (NaF, Na₃VO₄). Immunoprecipitationreactions were carried out using 1.5 mg of antibody-coupled beadsincubated with 100 mg of cell lysate at 4° C. for 30 minutes. Negativecontrols were immunoprecipitation reactions using beads coupled to IgGisotype control antibody (rabbit), and whole lysate fromimmunoprecipitation input was loaded directly onto a gel was used as apositive control.

Immunoblotting

Protein was collected by either standard whole-cell lysis using ice-coldradioimmunoprecipitation assay (RIPA) lysis buffer supplemented withprotease (Roche Life Scientific) and phosphatase inhibitors (NaF,Na₃VO₄) or by nuclear/cytosolic extraction using the NE-PER Nuclear andCytoplasmic Protein Extraction Reagent Kit according to themanufacturer's protocol (Pierce, Rockford, Ill., USA). Tissue proteinwas extracted by using a tissue grinder to lyse cells in 100 μL of RIPAlysis buffer for every 1 mg of tissue. Samples were centrifuged for 5minutes to remove depleted tissue debris. Protein concentration wasassessed with the Bradford assay. Protein lysates (30 ug/lane) wereresolved on a 10% SDS-PAGE denaturing gel, transferred to anitrocellulose membrane and blocked in 5% non-fat dry milk in 1× Trisbuffered saline supplemented with 0.01% Tween 20. Immunoblotting wasperformed by incubating membranes overnight at 4° C. using antibodies todetect 14-3-3ε, β, η, γ, σ, θ or ζ (Cell Signaling for all), P-Akt(S473) (Cell Signaling), total Akt (Cell Signaling), P-Bad (S136) (CellSignaling), total BAD (Cell Signaling) Survivin (Cell Signaling), MSIN3A(Abcam), GAPDH (Abcam) or α/β-tubulin (Cell Signaling). Bands weredetected using horseradish peroxidase-conjugated secondary antibodies(IgG mouse or rabbit) (Cell Signaling) and visualized using westernlightning plus chemiluminescent reagent (MidSci, Valley Park, Mo., USA)and signal read on a Chemidoc XRS Molecular Imager (Bio-RadLaboratories, Hercules, Calif., USA).

Caspase-3/7 Glo Assay

Apoptosis was assessed using a Caspase-3/7 Glo assay (PromegaCorporation, Madison, Wis., USA) in a 96-well white-bottom plate(BRANDplates, Wertheim, Germany) according to the manufacturer'sinstructions. Cells were seeded at a density of 8×10³ cells/well andincubated overnight before treatments or transfections. Luminescence wasread using a Cytation 5 multi-mode plate reader (BioTek Instruments,Winooski, Vt., USA).

Cycloheximide Assay

SCC12B.2 cells were plated at equal cell densities (5×10⁵ cells/plate)in 60 mm plates. Cells were first transfected with either control siRNA(Santa Cruz Biotechnology) or 14-3-3ε siRNA (Dharmacon). Forty-eighthours post-transfection the control siRNA transfected cells were treatedwith either vehicle (DMSO) or cycloheximide (100 μg/mL) (Abcam), while14-3-3ε siRNA transfected cells were treated with 100 μg/mLcycloheximide for 8 or 24 hours. To ensure equal numbers of cells wereused for immunoblotting, cells were collected by standardtrypsinization, resuspended in 500 μL of 1×PBS and counted on a CountessII automated cell counter (Thermo Fisher) four separate times to obtainan average cell number. Protein was collected by lysing 1.5×10⁶ cellsper treatment group. Equal volumes of cell lysate were used forimmunoblotting detection.

Mouse Tumor Xenografts

Groups of NCG mice (NOD CRISPR Prkdc Il2r gamma/NjuCrl) (Charles River,Malvern, Pa., USA) were injected s.c. with 5×10⁵ SCC13 cells suspendedin 1:1 mixture of 1×PBS:Matrigel (Corning, Corning, N.Y., USA). Once thetumors reached a volume of 150 mm³, tumors were treated with eithervehicle (50 μL 1×PBS) or 2.5 nmol/50 μL of the targeting peptide ES1P2.Tumors were injected directly with either vehicle or ES1P2 daily for 2days or every other day for 2 weeks and then harvested for analysis 24hours after the last treatment. Tumors were fixed overnight in 10%buffered formalin (Thermo Fisher) and then switched to 70% ethanol thefollowing day. All procedures were performed in accordance with theguidance of Creighton University Institutional Animal Care and UseCommittee.

14-3-3ε Conditional KO Mice Production

Conditional knockout of 14-3-3ε in the epidermis of the skin wasachieved by crossing 14-3-3ε^(fl/+) C57B16 mice with KRT14-Crerecombinase transgenic mice on an FVB/N background. Offspring werebackcrossed onto FVB/N mice for five generations to reduce backgroundheterogeneity. Genotyping for all mice was performed by polymerase chainreaction (PCR). To detect wild type and floxed alleles the followingprimers were used: aggtaccaaaacagtaagccatctcccta andgcatgtgtttgtctgtcagaggac. To detect the KO allele the following primerswere used: and ttcttttgtagaaattggggaaggtcatgg. All procedures wereperformed in accordance with the guidance of Creighton UniversityInstitutional Animal Care and Use Committee.

Assessment of Epidermal Hyperplasia

Epidermal hyperplasia was evaluated using hematoxylin and eosin(H&E)-stained skin sections from the ventral surface of each mouse. Theaverage epidermal thickness was determined by measuring from the outeredge of the most basal keratinocyte nucleus, to the edge of theoutermost keratinocyte nucleus at eight randomly-selected locationsalong the length of each skin section per mouse in a double-blindedmanner. The average number of epidermal cell layers was assessed bycounting the number of nucleated cell layers at eight randomly-selectedlocations in each section in a double-blinded manner. Measurements andcounts were done by using Olympus VS120 Slide Scanner Analysis Software(Olympus).

Chemical Carcinogenesis Protocol

At six weeks of age the dorsal hair of male and female 14-3-3ε WT and14-3-3ε MT mice was clipped (N=20 WT, N=18 MT). Two days after clipping,50 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) (50 nmol/200 μLacetone) was topically applied. Two weeks after DMBA treatment the micewere treated once weekly with 6.8 nmol of12-O-tetradecanoylphorbol-13-acetate (TPA) (6.8 nmol/200 μL in acetone)for 5 weeks. Following the five weeks of single TPA treatments, micewere treated 2 times per week with 6.8 nmol of TPA. Animals were weighed3 times per week and tumors were counted once per week. Groups of micewere euthanized at 24, 27 or 30 weeks. Paraffin-embedded sections ofeach tumor were assessed for histopathology by two board-certifieddermatologists. All procedures were performed in accordance with theguidance of the Creighton University Institutional Animal Care and UseCommittee.

Tetrapeptide Library

Three-dimensional structures for the virtual tetrapeptide library weregenerated using the YASARA program. The library contained twentystandard amino acid residues, where cysteine was excluded to preventsynthetic problems at later stage (19⁴=130,321 structures). Fourdifferent terminal end protection strategies were used in the library:free, N-acetyl, C-terminal amide and both termini capped(4×130,321=521,284 structures). A Python script was used to automatepeptide generation and minimization in vacuo. Minimization in solventwas not utilized since the torsional angles are randomized duringdocking: bond lengths and angles were possible candidates for correctionafter initial structure generation.

Target Selection and Docking

The N-terminal region with residue Asp6, Tyr9 and Gln6 of 14-3-3ε, asunique residues among of the seven isoforms of 14-3-3 proteins, wereidentified as a binding site. The non-protein components in the X-raystructure of 14-3-3ε were deleted and missing H-atoms were added usingYASARA. The resultant structure was relaxed in an aqueous environmentusing 200 ns Molecular Dynamics (MD) simulation. Binding site residueswere assigned using the ray-tracing algorithm included in the MolegroVirtual Docker (MVD) program. Receptor grids were aligned to thegeometric center of the identified binding sites and tetrapeptides weredocked using the GPU screening module of MVD. Hits were clustered usingTabu clustering of MVD with root mean square deviation (RMSD) cutoff of2.5 Å, ensuring maximum diversity in poses. Poses were ranked accordingto their molegro-derived energies; this scoring function is a modifiedversion of PLANTS.

Molecular Dynamics (MD) Simulation

For MD simulations, the GROMACS-2016 package was used with the CHARMM36mforce field. The structures of 14-3-3ε-peptide complex was solvated in adodecahedron with TIP3P water molecules and 0.15 M sodium chloride. Thesystem was subjected to 1000 steps steepest descent energy minimizationand then to 1 ns constant number of molecules, volume and temperature(NVT) simulation at 300 K so that the position of the protein-peptidecomplex was constrained at the center of the dodecahedron with a forceconstant of 1000 kJ·mol⁻¹. The system was then equilibrated during a 10ns unrestrained constant number of molecules, pressure and temperature(NPT) simulation at 1 bar pressure and 300 K temperature. Theintegration step was 2 fs, the LINCS algorithm was used to constrain allbonds to their correct length with a warning angle of 30°. Theprotein-peptide complex and the solvent with ions were coupled toseparate temperature baths with a relaxation constant of 0.1 ps. Thepeptide and the solvent with ions were coupled separately to constantpressure with a relaxation constant of 1.0 ps and 4.5×10⁻⁵ bar⁻¹isothermal compressibility. The temperature was controlled by astochastic velocity-rescaling method. The long-range electrostaticinteractions were calculated using the PME method with 1.2 nm cutoffdistance applying Verlet scheme and 0.15 nm Fourier spacing. For thecalculations of van der Waals interactions the short-range andlong-range cutoffs, respectively, were 1.0 and 1.2 nm using theforce-switch modifier. Finally, 250 ns NPT was performed at 300 K and 1bar pressure. The parameters were the same as during equilibrations,except the protein-peptide complex and solvent with ions were separatelycoupled to a 1 bar Parrinello-Rahman barostat. After the initial 250 nsscreening those structures in which the peptides did not dissociate fromthe Tyr9 region of 14-3-3ε were further simulated for 2 μs using thesame parameters as for the screening.

Residue Contribution Binding Energy

The residue contribution to the binding energy of peptides to 14-3-3εfor the last 50 ns of the 2 μs simulation was calculated using g_mmpbsasoftware. For the calculations solvent and solute dielectric constantwere 80 and 4, respectively. Temperature and sodium chlorideconcentration were the same as in the simulations.

Peptide Production

Peptides were synthesized and purified to greater that 95% purity byBachem (Bachem Americas Inc., Torrance, Calif., USA) and EZBiolabs(EZBiolabs, Carmel, Ind., USA).

Neutral Red Cell Viability Assay

To determine the half maximal inhibitory concentration (IC₅₀) for ES1P2in SCC12B.2 and nHEK cells, cells were plated in a 96-well plate at adensity of 6×10³ cells/well. Twenty-four hours after plating, cells weretreated with increasing concentrations of ES1P2 (0-75 μM) in Opti MEMmedium supplemented with reduced serum (Invitrogen) for 4 consecutivedays. ES1P2 peptide was resuspended fresh, daily in 25 mM Tris buffer(pH 7.5). Neutral Red Cell Viability assays were performed, whereabsorbance was measured using a Cytation 5 multi-mode plate reader(BioTek). The experiment was repeated three separate times with 4replicate wells per experiment to obtain an average IC₅₀ value.

TUNEL Assay

TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) wascarried out using Promega DeadEnd Fluorometric TUNEL system inaccordance with the manufacturer's instructions (Promega). Cells weretreated with DAPI (Vector Labs) to locate the nuclei within cells.TUNEL-positive cells were those that displayed a clear nuclear signal.Slides were digitalized and apoptotic nuclei counted using an OlympusVS120 Slide Scanner (Olympus).

Statistical Analysis

Comparisons between two groups was performed using a two-tailedStudent's t-test, where P≤0.05. Significance was determined usingone-way ANOVA with the Dunnett's post-hoc test for comparison of morethan two groups, where P≤0.05. For data collected at multiple timepoints, two-way ANOVA with a Bonferroni correction was used, whereP≤0.05.

Methods and Materials—pS and pT Targeting Peptide Implementations

Cell Culture

Carcinoma human cell lines (SCC12B.2 and SCC13) were maintained inDulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, Calif.,USA) supplemented with 1% penicillin (10 000 U/ml)-streptomycin (10 000μg/ml) (PenStrep) (Invitrogen) and 10% fetal bovine serum (GeminiBio-Products, Sacramento, Calif., USA) at 37° C. and 5% CO2. Primarynormal human epidermal keratinocytes (nHEK) (Thermo Fisher Scientific,Waltham, Mass., USA) isolated from neonatal foreskin were maintained inEpiLife medium (Invitrogen) supplemented with human keratinocyte growthsupplement (HKGS) and 1% PenStrep. nHEK cells were not passaged morethan twice before experiments. Cells were treated with CDC25A inhibitorNSC663284 (Tocris Bioscience, Bristol, United Kingdom) suspended inDMSO, transfected with wild type CDC25A or control plasmid usingLipofectamine/Plus transfection reagents (Life Technologies, Carlsbad,Calif., USA) or transfected with siRNA targeting 14-3-3 ε or controlsiRNA (Santa Cruz Biotechnology, Dallas, Tex., USA) using siQuesttransfection reagents (Mirus Bio, Madison, Wis., USA).

Peptide Structure and Molecular Dynamics Simulations

The pS and pT peptides include the 173-186 and 501-515 fragments ofhuman CDC25A, respectively, and were digitally generated by the YASARAprogram. Peptide pS further includes the phospho-Ser178 (pSer) and pTfurther includes the phospho-Thr507 (pThr) residue. Residues of anexisting peptide in the X-ray structure of 14-3-3ε were mutated to thecorresponding CDC25A fragments residues. Both peptides were N-terminallyacetyl- and C-terminally amide-protected to preserve the electronicstructure of the backbone. The resultant structure was energy-minimizedin gas phase using two steps: 1) the atoms of 14-3-3ε were frozen andthe ligand peptide was relaxed; and 2) in a subsequent step the wholesystem was relaxed. Stability of the prepared peptide-14-3-3ε complexeswas determined by molecular dynamics (MD) simulations using theGROMACS-2016 package and the CHARMM36m force field.

The structures of the 14-3-3ε-peptide complexes were solvated in adodecahedron with TIP3P water molecules and 0.15 M sodium chloride. Thesystem was subjected to 1000 steps steepest descent energy minimizationand then to 1 ns constant number of molecules, volume and temperature(NVT) simulation at 300 K so that the position of the protein-peptidecomplex was constrained at the center of the dodecahedron with a forceconstant of 1000 kJ·mol⁻¹. The system was then equilibrated during 10 nsunrestrained constant number of molecules, pressure and temperature(NPT) simulation at 1 bar pressure and 300 K temperature. Theintegration step was 2 fs, the LINCS algorithm was used to constrain allbonds to their correct length with a warning angle of 30°. The peptideand the solvent with ions were coupled to separate temperature bathswith a relaxation constant of 0.1 ps. The system was coupled to constantpressure with a relaxation constant of 1.0 ps and 4.5×10⁻⁵ bar⁻¹isothermal compressibility. The temperature was controlled by astochastic velocity-rescaling method. The long-range electrostaticinteractions were calculated using the PME method with 1.2 nm cutoffdistance applying the Verlet scheme and 0.15 nm Fourier spacing. For thecalculations of van der Waals interactions the short-range andlong-range cutoffs, respectively, were 1.0 and 1.2 nm using theforce-switch modifier. Finally, 1000 ns NPT simulation was performed at300 K and 1 bar pressure. The parameters were the same as duringequilibration, except that the temperature was kept constant by usingleap-frog stochastic dynamics integrator with τ_(T) of 2 ps and thesystem was coupled to 1 bar Parrinello-Rahman barostat.

Trajectory Analysis

Configurational entropy was calculated to determine if thermodynamicequilibrium was reached. The covariance matrix for the Ca-atoms wascalculated using the covar module of GROMACS, the eigenvectorscorresponding to the 150 highest eigenvalues were used to calculate thebackbone configurational entropy. Stability of the protein-peptidecomplex was determined by calculating the root-mean-square deviations(RMSD) of the Ca-atoms. Representative structures of the simulation weredetermined by using the GROMOS method of clustering with Ca-atoms RMSDcutoff of 0.2 nm using structures sampled in 100 ps intervals.

Peptide Production

Peptides were synthesized and purified to greater than 95% purity byBachem (Bachem Americas Inc., Torrance, Calif., USA) and EZBiolabs(EZBiolabs, Carmel, Ind., USA).

Immunofluorescence

Immunostaining was performed using antibodies recognizing P-Akt (S473)(Thermo Fisher) and Survivin (Cell Signaling, Danvers, Mass., USA), withalexa-fluor488-conjugated secondary antibody (IgG rabbit) (Invitrogen)with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame,Calif., USA) to identify the nuclei. Slides were digitalized andanalyzed using an Olympus VS120 Slide Scanner (Olympus, Shinjuku, Tokyo,JPN).

Co-Immunoprecipitation

Co-Immunoprecipitation (Co-IP) was performed using a Dynabeads Protein GImmunoprecipitation kit (Life Technologies, Carlsbad, Calif., USA).Beads were coupled to antibodies targeting 14-3-3ε (Fisher Scientific,Waltham, Mass., USA) or CDC25A (Santa Cruz Biotechnology, Dallas, Tex.,USA) at a ratio of 7 μg of antibody for every 1 mg of beads according tothe manufacturers protocol. SCC12B.2 cells were treated with vehicle (20mM Tris buffer, pH 7.5) or fresh peptide (pS or pT) daily at the IC₅₀concentration for 48 hours in Opti-MEM I Reduced Serum Medium(Invitrogen) and collected by trypsinization. One hundred mg of cellswere lysed in 1×IP buffer supplemented with 100 mM sodium chloride, 2 mMdithiothreitol, 1 mM magnesium chloride and protease/phosphataseinhibitors. Immunoprecipitation reactions were carried out using 1.5 mgof antibody-coupled beads incubated with 100 mg of cell lysate at 4° C.for 30 minutes. Negative controls were immunoprecipitation reactionsusing beads coupled to IgG isotype control antibody (rabbit), and wholelysate from immunoprecipitation input was loaded directly onto a gel wasused as a positive control.

Immunoblotting

Protein was collected by either standard whole-cell lysis using ice-coldradioimmunoprecipitation assay (RIPA) lysis buffer supplemented withprotease (Roche Life Scientific) and phosphatase inhibitors (NaF,Na₃VO₄) or by nuclear/cytosolic extraction using the NE-PER Nuclear andCytoplasmic Protein Extraction Reagent Kit according to themanufacturer's protocol (Pierce, Rockford, Ill., USA). Tissue proteinwas extracted by using a tissue grinder to lyse cells in 100 μL of RIPAlysis buffer for every 1 mg of tissue. Samples were centrifuged for 5minutes to remove depleted tissue debris. Protein concentration wasassessed with the Bradford assay. Protein lysates (30 ug/lane) wereresolved on a 10% SDS-PAGE denaturing gel, transferred to anitrocellulose membrane and blocked in 5% non-fat dry milk in 1× Trisbuffered saline supplemented with 0.01% Tween 20. Immunoblotting wasperformed by incubating membranes overnight at 4° C. using antibodies todetect 14-3-3ε, β, η, γ, σ, θ or ζ (Cell Signaling for all), CDC25A(Santa Cruz Biotechnology), P-Akt (S473) (Cell Signaling), total Akt(Cell Signaling), P-Bad (S136) (Cell Signaling), total BAD (CellSignaling), Survivin (Cell Signaling), or GAPDH (Cell Signaling). Bandswere detected using horseradish peroxidase-conjugated secondaryantibodies (IgG mouse or rabbit) (Cell Signaling) and visualized usingwestern lightning plus chemiluminescent reagent (MidSci, Valley Park,Mo., USA) and signal read on a Chemidoc XRS Molecular Imager (Bio-RadLaboratories, Hercules, Calif., USA).

Apoptosis Antibody Array

Protein was extracted from SCC12B.2 cells using lysis buffersupplemented with protease and phosphatase inhibitors. SCC12B.2 cellswere transfected with empty vector DNA or wild-type CDC25A for 48 hours,or control (Santa Cruz Biotechnology) or 14-3-3ε siRNA for 48 hours(Dharmacon). Array membranes (Cell Signaling), containing antibodiesagainst 19 pro- and anti-apoptotic proteins were incubated withwhole-cell lysates overnight at 4° C. according to the manufacturer'sinstructions. Immunoreactive dots were visualized using westernlightning plus chemiluminescent detection system (MidSci) to generate asignal on a Chemidoc XRS Molecular Imager (Bio-Rad Laboratories).

Mouse Tumor Xenografts

Groups of NCG mice (NOD CRISPR Prkdc Il2r gamma/NjuCrl) (Charles River,Malvern, Pa., USA) were injected s.c. with 5×10⁵ human SCC13 cellssuspended in 1:1 mixture of 1×PBS:Matrigel (Corning, Corning, N.Y.,USA). Once the tumors reached 150 mm³ in volume they were treatedintratumorally with either vehicle (1× sterile PBS, pH, 7.5), pS, or pT(2.5 nmol/50 μL for each) daily for 2 days and then the mice wereeuthanized 24 hours after the second treatment. Tumors were fixedovernight in 10% neutral buffered formalin (Thermo Fisher) and thenswitched to 70% ethanol the following day before embedding andsectioning. All procedures were performed in accordance with theguidance of the Creighton University Institutional Animal Care and UseCommittee.

TUNEL Assay

TUNEL (terminal uridine nick-end labelling) assays were carried outusing the Promega DeadEnd Fluorometric TUNEL system in accordance withthe manufacturer's instructions (Promega Corporation, Madison, Wis.,USA). Cells were treated with 4′,6-diamidino-2-phenylindole (DAPI) tolocate the nuclei within cells. Slides were scanned and analyzed usingan Olympus VS120 Slide Scanner (Olympus).

Caspase-3/7 Glo Assay

Apoptosis was assessed using a Caspase-3/7 Glo assay (Promega) in a96-well white-bottom plate (BRANDplates, Wertheim, Germany). Cells wereseeded at a density of 8×10³ cells/well and Caspase-3/7 glo wasperformed 24 hours post-treatment according to the manufacturer'sinstructions and read using a Cytation 5 multi-mode plate reader(BioTek, Instruments, Winooski, Vt., USA).

Neutral Red Cell Viability Assay

To determine the half maximal inhibitory concentration (IC₅₀) for pS andpT in SCC12B.2 cells, cells were plated in a 96-well plate at a densityof 6×10³ cells/well. Twenty-four hours after plating cells were treatedwith increasing concentrations of pS and pT peptides (0-75 μM) in OptiMEM media supplemented with reduced serum (Invitrogen) for 4 consecutivedays. The peptides were resuspended fresh daily in 20 mM Tris buffer (pH7.5). Neutral Red Cell Viability assays were performed, where absorbancewas measured using a Cytation 5 multi-mode plate reader (BioTek). Theexperiment was repeated three separate times with 4 replicate wells ineach experiment to obtain an average IC₅₀ value.

Statistical Analysis

Significance was determined using one-way ANOVA with the Dunnett'spost-hoc test for comparison of more than two groups, where P≤0.05.Comparisons between two groups were performed using a two-tailedStudent's t-test, where P≤0.05.

Differential Scanning Fluorimetry Analysis

Differential scanning fluorimetry (DSF) experiments were performed usingBioRad CFX384 Real-Time qPCR attached with C1000 Touch Thermo Cycler inthe FRET channel. 7 μM subunits of 14-3-3ε protein, ten molar excess ofSypro Orange and 20 mM Tris-HCl, pH 7.4 assay buffer were used in allthe melting experiments. Stock solution of peptides were prepared bydissolving 1 mg of lyophilized peptides in 20 mM Tris, pH 7.4 buffer and0.1 μM to 200 μM dilution series were prepared. Protein meltingexperiments were performed both in the absence and the presence ofincreasing concentration of peptides. 15 μL of each samples (intriplicate) were put into each well of a 384-well white PCR plate andsealed with optical adhesive sealer (BioRad MSB-1001) and any air-bubblein the solutions were removed by spinning the plate vertically with PCRplate spinner (Labnet MPS 1000) at 1000 RPM for 3 minutes. Thetemperature was increased from 25° C. to 95° C. at a rate of 0.7° C. perminute. Melting temperatures of each well were determined using the CFXMaestro software version 2.0 by plotting the first derivative offluorescence signal against temperature. All the measurements wereperformed in triplicate and the deviations of melting temperatures (ΔTm)presented for each samples were calculated relatively to three controlsamples (7 μM 14-3-3ε in the absence of the peptide). Binding constant(K_(d)) for each peptide was calculated by plotting ΔTm against peptideconcentration and data points were fitted with Hill's equation usingGraphPad 9.0 software.

FIG. 4A shows differential scanning fluorimetry (DSF) results of bindingaffinity to 14-3-3ε protein of the third targeting peptide (SEQ ID NO:3), which had a K_(d) of 3.64 μM. FIG. 4B shows DSF results of bindingaffinity to 14-3-3ε protein of a targeting peptide that shortened thepeptide from FIG. 4A at the C-terminus, which resulted in a K_(d) of2.16 μM, showing non-significant change in K_(d) as compared to the DSFresults from FIG. 4A. FIG. 2C shows that a Tyr substitution in thesequence of the peptide from FIG. 4A (resulting in a sequence ofAc-Arg-Thr-Lys-Ser-Arg-Thr(PO₃ ²⁻)-Tyr-Ala-Gly-NH₂) was well toleratedwith a K_(d) of 2.34 μM. FIG. 4D shows DSF results of binding affinityto 14-3-3ε protein of the second targeting peptide (SEQ ID NO: 2), whichhad a K_(d) of 11.23 μM, which was approximately three times lower thanthat of the peptide from FIG. 4A. Shortening of the peptide from FIG. 4Dat both the N- and C-terminus decreased its binding affinity to 20.6 μM,as shown in FIG. 4E, but a Tyr substitution (resulting in a sequence ofAc-Gln-Arg-Tyr-Asn-Ser-(PO₃ ²⁻)-Ala-Pro-Ala-Arg-NH₂) and a Phesubstitution (resulting in a sequent of Ac-Gln-Arg-Phe-Asn-Ser-(PO₃²⁻)-Ala-Pro-Ala-Arg-NH₂) substantially improved the binding affinity ofthe peptide from FIG. 4D with a K_(d) of 9.34 μM and a K_(d) of 8.29 μM,respectively, as shown in FIGS. 4F and 4G.

CONCLUSION

Although the subject matter has been described in language specific tostructural features and/or process operations, it is to be understoodthat the subject matter defined in the appended claims is notnecessarily limited to the specific features or acts described above.Rather, the specific features and acts described above are disclosed asexample forms of implementing the claims.

The invention claimed is:
 1. A peptide composition, the peptidecomposition having a peptide consisting of the formula:X—R¹—R²—R³—R⁴-Ser-(PO₃ ²⁻)—R⁵-Pro-R⁶-Arg-Y, wherein X representshydrogen, acetyl or propionyl group, R¹ represents an L or D-amino acidresidue selected from the group consisting of: Gln, Trp, Tyr, Phe, His,R² represents an L or D-amino acid residue selected from the groupconsisting of: Arg, Lys, Orn, R³ represents an L or D-amino acid residueselected from the group consisting of: Gln, Arg, Lys, Orn, Pro, Trp,Tyr, Phe, His, R⁴ represents an L or D-amino acid residue selected fromthe group consisting of: Asn, Arg, Lys, Orn, Trp, Tyr, Phe, His, R⁵represents an L or D-amino acid residue selected from the groupconsisting of: Ala, Trp, Tyr, Phe, His, R⁶ represents an L or D-aminoacid residue selected from the group consisting of: Ala, Trp, Tyr, Phe,His, and Y represents —OH, amide, methylamide, or ethylamide groups. 2.The peptide composition of claim 1, wherein the peptide promotesapoptosis in squamous cell carcinoma cells.
 3. The peptide compositionof claim 1, further comprising a pharmaceutically acceptable carrierselected from the group consisting of an emulsion, a lotion, a cream, agel, a water-immiscible solvent, an emollient, and combinations thereof.4. The peptide composition of claim 1, further comprising apharmaceutically acceptable carrier suitable for administration selectedfrom the group consisting of injection, topical, aerosol, inhalation,oral, systemic IV, ocular, and rectal.
 5. A method of reducing squamouscell carcinoma survival comprising contacting a squamous cell carcinomacell with the peptide composition of claim 1.